Biological and Mechanical Evaluation of Novel Prototype Dental Composites.

The breakdown of the polymeric component of contemporary composite dental restorative materials compromises their longevity, while leachable compounds from these materials have cellular consequences. Thus, a new generation of composite materials needed to be designed to have a longer service life and ensure that any leachable compounds are not harmful to appropriate cell lines. To accomplish this, we have developed concurrent thiol-ene-based polymerization and allyl sulfide-based addition-fragmentation chain transfer chemistries to afford cross-linked polymeric resins that demonstrate low shrinkage and low shrinkage stress. In the past, the filler used in dental composites mainly consisted of glass, which is biologically inert. In several of our prototype composites, we introduced fluorapatite (FA) crystals, which resemble enamel crystals and are bioactive. These novel prototype composites were benchmarked against similarly filled methacrylate-based bisphenol A diglycidyl ether dimethacrylate / triethylene glycol dimethacrylate (bisGMA/TEGDMA) composite for their cytotoxicity, mechanical properties, biofilm formation, and fluoride release. The leachables at pH 7 from all the composites were nontoxic to dental pulp stem cells. There was a trend toward an increase in total toughness of the glass-only-filled prototype composites as compared with the similarly filled bisGMA/TEGDMA composite. Other mechanical properties of the glass-only-filled prototype composites were comparable to the similarly filled bisGMA/TEGDMA composite. Incorporation of the FA reduced the mechanical properties of the prototype and bisGMA/TEGDMA composite. Biofilm mass and colony-forming units per milliliter were reduced on the glass-only-filled prototype composites as compared with the glass-only-filled bisGMA/TEGDMA composite and were significantly reduced by the addition of FA to all composites. Fluoride release at pH 7 was greatest after 24 h for the bisGMA/TEGDMA glass + FA composite as compared with the similarly filled prototypes, but overall the F- release was marginal and not at a concentration to affect bacterial metabolism.

Signals in Stem Cell Differentiation on Fluorapatite-Modified Scaffolds.

Previously, we reported that the fluorapatite (FA)-modified polycaprolactone (PCL) nanofiber could be an odontogenic/osteogenic inductive tissue-engineering scaffold by inducing stem cell differentiation and mineralization. The present study aimed to explore which of the signal pathways affected this differentiation and mineralization process. The Human Signal Transduction PathwayFinder RT2 Profiler PCR Array was used to analyze the involvement of potential signal transduction pathways during human dental pulp stem cell (DPSCs) osteogenic differentiation induced by FA-modified PCL nanofiber scaffolds. Based on the results, perturbation studies of the signaling pathways hedgehog, insulin, and Wnt were performed. Moreover, the autophagy process was studied, as indicated by the expression of the microtubule-associated protein 1 light chain 3A/B-II (LC3-II) and the cell osteogenic phenotypic changes. In a comparison of the cells grown on PCL + FA scaffolds and those on PCL-only scaffolds, the transcript expression of BMP2, BMP4, FOXA2, PTCH1, WNT1, and WNT2 (PCR array-labeled signal proteins of the hedgehog pathway); CEBPB, FASN, and HK2 (PCR array-labeled signal proteins of the insulin pathway); and CCND1, JUN, MYC, TCF7, and WISP1 (PCR array-labeled signal proteins of the Wnt pathway) doubled at day 14 when obvious cell osteogenic differentiation occurred. Phenotypically, in all the perturbation groups at day 14, ALP activity, OPN, and autophagy marker LC3-II expression were coincidently decreased. Consistently, no positive alizarin red staining or von Kossa staining was observed in the specimens from these perturbation groups at day 28. The results showed that when obvious cell differentiation occurred at day 14 on PCL + FA control groups, the inhibition of the hedgehog, insulin, and Wnt pathways significantly decreased DPSC osteogenic differentiation and mineralization. The osteogenic differentiation of DPSCs grown on FA-modified PCL scaffolds appeared to be positively modulated by the hedgehog, insulin, and Wnt signal pathways, which were coordinated with and/or mediated by the cell autophagy process.

Autophagy Modulates Cell Mineralization on Fluorapatite-Modified Scaffolds.

As a major intracellular degradation and recycling machinery, autophagy plays an important role in maintaining cellular homeostasis and remodeling during normal development. Our previous study showed that fluorapatite (FA) crystal-coated electrospun polycaprolactone (PCL) was capable of inducing differentiation and mineralization of human dental pulp stem cells. However, how autophagy changes and whether autophagy plays a vital role during these processes is still unknown. In this study, we seeded STEMPRO human adipose-derived stem cells (ASCs) on both PCL+FA and PCL scaffolds to investigate the osteogenic inductive ability of FA crystals and we observed the autophagy changes of these cells. Scanning electron microscopy and fluorescence microscopy images, along with DNA quantitation, showed that both PCL+FA and PCL scaffolds could sustain ASC growth but only the PCL+FA scaffold could sustain cell mineralization. This was confirmed by alkaline phosphatase activity and Alizarin red and Von Kossa staining results. The autophagy RT2 Profiler polymerase chain reaction array analysis showed many autophagy-related genes changes during ASC differentiation. Western blot analysis indicated that several autophagy-related proteins fluctuated during the procedure. Among them, the microtubule-associated protein 1 light chain 3 (LC3)-II protein changes of the ASCs grown on the 2- or 3-dimensional environments at 6 h, 12 h, 1 d, 3 d, 7 d, 14 d, and 21 d reached a peak value at day 7 during osteogenesis. At earlier stages (from day 0 to day 3), the addition of autophagy inhibitors (3-mathyladenine, bafilomycin A1, and NH4Cl) attenuated the expression of osteogenic related markers (osteopontin, alkaline phosphatase activity, Alizarin red, and Von Kossa) compared with the control group. All data indicated that autophagy played an important role in ASC differentiation on the PCL+FA scaffold. Inhibition of autophagy before day 3 strongly inhibited osteogenic differentiation and mineralization of ASCs in the 3-dimensional model. This observation further elucidates the mechanism of autophagy in mesenchymal stem cell osteogenic differentiation.

Noninvasive dentistry: a dream or reality?

Various caries prevention and repair strategies are reviewed in this article ranging from the use of fluoride to nanohydroxyapatite particles. Several of the strategies which combine fluoride and calcium and phosphate treatments have both in vitro and in vivo data showing them to be efficacious if the surface integrity of the lesion is not breached. Once this has occurred, the rationale for cutting off the nutrient supplies to the pathogenic bacteria without the removal of the infected dentine, a noninvasive restorative technique, is discussed using existing clinical studies as examples. Finally two novel noninvasive restorative techniques using fluorohydroxyapatite crystals are described. The need for clinical data in support of emerging caries-preventive and restorative strategies is emphasized.

VEGF promotes osteogenic differentiation of ASCs on ordered fluorapatite surfaces.

Vascular endothelial growth factor (VEGF) has been reported to mediate both osteogenesis and angiogenesis in bone regeneration. We previously found an upregulation of VEGF in adipose-derived stem cells (ASCs) when obvious mineralization occurred on a novel fluorapatite (FA)-coated surfaces. This study investigated the effect of FA and VEGF on the growth, differentiation and mineralization of (ASC) grown on ordered FA surfaces. Cells grown on FA and treated with VEGF demonstrated osteogenic differentiation as measured with ALP staining, and obvious mineralization as measured by Alizarin red staining. A combined stimulating effect of FA and VEGF was seen using both indicators. VEGF signaling pathway perturbation using a specific VEGF receptor inhibitor showed the lowest levels of ALP and Alizarin red staining, which was partially rescued when the cells were grown on FA and/or treated with the addition of VEGF. The osteogenic differentiation of ASCs stimulated by these FA surfaces as well as VEGF has been shown to be mediated through, but probably not only, the VEGF signaling pathway. The enhancement of osteogenic differentiation and mineralization supports the potential use of therapeutic VEGF and FA coatings in bone regeneration.

Fluorapatite-modified scaffold on dental pulp stem cell mineralization.

In previous studies, fluorapatite (FA) crystal-coated surfaces have been shown to stimulate the differentiation and mineralization of human dental pulp stem cells (DPSCs) in two-dimensional cell culture. However, whether the FA surface can recapitulate these properties in three-dimensional culture is still unknown. This study examined the differences in behavior of human DPSCs cultured on electrospun polycaprolactone (PCL) NanoECM nanofibers with or without the FA crystals. Under near-physiologic conditions, the FA crystals were synthesized on the PCL nanofiber scaffolds. The FA crystals were evenly distributed on the scaffolds. DPSCs were cultured on the PCL+FA or the PCL scaffolds for up to 28 days. Scanning electron microscope images showed that DPSCs attached well to both scaffolds after the initial seeding. However, it appeared that more multicellular aggregates formed on the PCL+FA scaffolds. After 14 days, the cell proliferation on the PCL+FA was slower than that on the PCL-only scaffolds. Interestingly, even without any induction of mineralization, from day 7, the upregulation of several pro-osteogenic molecules (dmp1, dspp, runx2, ocn, spp1, col1a1) was detected in cells seeded on the PCL+FA scaffolds. A significant increase in alkaline phosphatase activity was also seen on FA-coated scaffolds compared with the PCL-only scaffolds at days 14 and 21. At the protein level, osteocalcin expression was induced only in the DPSCs on the PCL+FA surfaces at day 21 and then significantly enhanced at day 28. A similar pattern was observed in those specimens stained with Alizarin red and Von Kossa after 21 and 28 days. These data suggest that the incorporation of FA crystals within the three-dimensional PCL nanofiber scaffolds provided a favorable extracellular matrix microenvironment for the growth, differentiation, and mineralization of human DPSCs. This FA-modified PCL nanofiber scaffold shows promising potential for future bone, dental, and orthopedic regenerative applications.

In vitro anti-caries effect of fluoridated hydroxyapatite-coated preformed metal crowns.

AIM: To synthesise fluoridated hydroxyapatite (FA) crystals directly on preformed metal crowns (PMCs) and evaluate the anti-cariogenic properties in an in vitro model. METHODS: FA crystals were grown on etched PMCs and stainless steel discs and characterised by SEM. FA-coated discs allowed fluoride release to be assessed from a known surface area of FA crystals. Discs were divided into four groups (n = 6/group) and exposed to solutions at pH 4-7. Fluoride levels in solution were measured after each exposure. Twelve FA-coated and 12 non-coated PMCs were cemented onto human molars using glass ionomer (GI) or unfilled resin, making four groups of six teeth; FA-coated + GI, FA-coated + resin; non-coated + GI and non-coated + resin. Teeth were exposed to acidified gelatin (pH = 4.3) for 9 weeks. RESULTS: SEM showed FA crystal growth on interior and exterior of the crowns. Average fluoride release from FA-coated discs was 0.16 mg/L/cm² at pH < 5.0. Teeth were sectioned through the lesion. Polarised microscopic examination revealed significantly smaller lesions in FA-coated crown groups compared to non-coated crown groups. CONCLUSION: FA-coated PMCs demonstrated carious lesion preventing effects, i.e. fluoride release and reduction of demineralisation at crown/tooth interface. FA-coated crowns could be an aesthetic, inexpensive and caries preventive alternative in clinical dentistry.

Adhesion and growth of dental pulp stem cells on enamel-like fluorapatite surfaces.

UNLABELLED: To study how apatite crystal alignment of an enamel-like substrate affects DPSC cellular adhesion and growth as a precursor to produce an in vitro enamel/dentin superstructure for future studies. The cells were subcultured in 10% FBS DMEM up to seven weeks on the two surfaces. Specimens were observed under SEM, counted, and analyzed using the human pathway-focused matrix and adhesion PCR array. After three days, the cell number on ordered FA surface was significantly higher than on the disordered surface. Of the 84 focused pathway genes, a total of 20 genes were either up or down regulated in the cells on ordered FA surface compared to the disordered surface. More interestingly, of the cell-matrix adhesion molecules, integrin alpha 7 and 8 (ITGA 7 and 8), integrin beta 3 and 4 (ITGB3 and 4), and the vitronectin receptor-integrin alpha V (ITGAV) and the key adhesion protein-fibronectin1 (FN1) were up-regulated. In SEM, both surfaces showed good biocompatibility and supported long term growth of DPSC cells but with functional cell-matrix interaction on the ordered FA surfaces. SIGNIFICANCE: The enhanced cellular response of DPSC cell to the ordered FA crystal surface involves a set of delicately regulated matrix and adhesion molecules which could be manipulated by treating the cells with a dentin extract, to produce a dentin/enamel superstructure.

Effects of systemic fluoride and in vitro fluoride treatment on enamel crystals.

Systemically administered fluoride at a concentration of 75 ppm increases the surface roughness of developing enamel crystals in rats, which may be significant in advancing our understanding of the biological mechanism of fluorosis. Thus, the aim of this study was to investigate whether the increased surface roughness may be a result of surface restructuring by the direct action of fluoride at the crystal surface. We examined the fluoride dose-dependent roughening of enamel crystal surfaces in vivo, in the rat, and whether this roughening could be mimicked by the in vitro treatment of rat enamel crystals with neutral pH fluoride solutions. Our results showed that enamel crystal surface roughness increased after treatment with increasing fluoride ion concentrations, whether applied in vitro or administered systemically. This suggests a mechanism, alongside others, for the increased surface roughness of crystals in fluorotic enamel.

Dentin-pulp complex responses to carious lesions.

To understand the molecular events underlying the dentin-pulp complex responses to carious progression, we systematically analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries. Dentin matrix proteins analyzed included collagen type I, phosphophoryn (PP) and dentin sialoprotein (DSP), all of which play decisive roles in the dentin mineralization process. Human non-carious and carious third molar teeth were freshly collected, demineralized, and processed for hematoxylin and eosin staining. The ABC-peroxidase method was used for immunohistochemical staining of collagen type I, PP and DSP proteins using specific antibodies. In situ hybridization was also performed. In contrast to elongated odontoblasts in non-carious teeth, odontoblasts subjacent to dentin caries were cuboidal and fewer in number. The predentin zone was also dramatically reduced in teeth with dentin caries. The staining intensity for collagen type I, PP and DSP in the dentin-pulp complex increased progressively from non-carious teeth, to teeth with enamel and dentin caries. In situ hybridization studies showed DSP-PP mRNA expression in odontoblasts and dental pulp that was consistent with our immunohistochemical results. These results suggest that carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type I, PP and DSP proteins. This response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation.

Interaction of dendrimers (artificial proteins) with biological hydroxyapatite crystals.

This investigation sets out to mimic protein-crystal interaction during biomineralization with the use of artificial proteins (dendrimers). It is hypothesized that these interactions depend on the surface charge of hydroxyapatite crystals. This was investigated with the use of dendrimers with capped surfaces of different charges to probe the surface. We used AFM images of crystal-bound dendrimers to determine the distribution of the surface charge, and its magnitude was correlated to the binding capacity of the dendrimers to the surface. The binding capacity of the dendrimers in ascending order at pH 7.4 was: acetamide-capped, -NHC(O)CH3, neutral charge; carboxylic-acid-capped, -COOH, negative charge; and amine-capped, -NH2, positive charge. AFM images of the crystals showed dendrimers spaced equally along the crystal. The results suggest that the crystal surface has alternating bands of positive and negative charge or a differential charge array, i.e., alternating bands of either more or less positive or negative charge.

Binding of dentin noncollagenous matrix proteins to biological mineral crystals: an atomic force microscopy study.

Noncollagenous matrix proteins (NCPs) of dental hard tissues (dentin, cementum) are involved, both temporally and spatially, in the mineralization of their collagen matrices. Two of the NCPs thought to initiate mineral nucleation and control crystal growth in dentin, are dentin phosphoproteins (DPP) and dentin sialoprotein (DSP). Control of crystal growth would depend on the binding capacity of these two molecules, which may be related to the charge domains on the crystals and/or the phosphorylation of the protein. Phosphophoryn (a highly phosphorylated DPP) and DSP were isolated, purified, and characterized from the immature root apicies of human teeth. Dephosphorylation of phosphophoryn was carried out using bovine intestinal alkaline phosphatase. Enamel crystals were prepared from the maturation stage of developing rat incisor enamel. Protein-coated crystals were prepared for viewing in an atomic force microscope fluid cell using tapping mode. Desorption of the proteins was achieved using a phosphate buffer and surface roughness measurements were obtained from all specimens. Time-lapsed images of the crystals showed "nanospheres" of protein distributed along the crystals but only the phosphophoryn-coated crystals showed a distinctive banding pattern, which was still visible after the phosphate desorption experiments. The surface roughness measurements were statistically greater (P <0.01) when compared to the control for only the phosphophoryn-coated specimens. It is hypothesized that the phosphophoryn binding may be associated with charge arrays on the crystal surface and its phosphorylation. Also, based on its affinity for the crystalsurfaces, phosphophoryn seems the most likely candidate for controlling dentin crystal growth and morphology.

Emerging methods used in the prevention and repair of carious tissues.

A systematic review was undertaken to investigate four emerging methodologies that might be used in the prevention of caries and/or repair of carious tissues. These included a partitioned dentifrice, laser technology, fluoride-releasing dental materials, and for deep carious lesions, bone morphogenic protein (BMP) therapy. The search strategy was to review articles written in English, indexed in MEDLINE and EMBASE databases and published since 1976. Over two hundred articles were read but because of the inclusion and exclusion criteria, only thirty-three were included in the evidence tables. The review of partitioned toothpaste showed either a greater remineralizing effect or a greater increase in the resistance to demineralization of both enamel and dentin, with the exception of its lack of effectiveness on coronal caries in the only clinical trial. Five of the six in vitro studies on enamel and the one study on dentin reported that lased tissue was less soluble than nonlased. Six clinical and four in situ studies were reviewed in answering the question as to whether fluoride-releasing restorative materials increase the remineralization or resistance to demineralization of human enamel and dentin. Eight of these reported positive findings. Six animal studies investigating BMP were reviewed, and all showed the ability of BMP to induce tubular dentin formation. Although the laboratory, animal, and limited clinical trials report encouraging results, independent, randomized, controlled clinical trials need to be carried out before these emerging technologies can be recommended for use in general practice.

Molecular cloning of a human dentin sialophosphoprotein gene.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP; phosphophoryn) are two principal dentin-specific non-collagenous proteins. DPP is extremely acidic and is rich in aspartic acid and serine. By virtue of this structure, DPP may bind large amounts of calcium and may facilitate initial mineralization of dentin matrix collagen as well as regulate the size and shape of the crystals. The function of DSP is not known. DSP and DPP are encoded by a single gene in both rat and mouse, and are uniquely expressed in odontoblasts and transiently in pre-ameloblasts. Because DSP and DPP are isolated from dentin as distinct proteins and appear to be present in different amounts, the nascent dentin sialophosphoprotein (DSPP) is likely cleaved to yield DSP and DPP. However, when, where and how the DSPP is cleaved into DSP and DPP is not clear. To further elucidate the structure and function of human DSP and DPP, we have cloned DPP and DSP cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) strategies, and then cloned and initiated characterization of a human dentin sialophosphoprotein gene. The genomic organization of human DSPP is very similar to that of mouse, containing five exons and four introns, suggesting it is a homologue of mouse dentin sialophosphoprotein (DSPP). Exons 1-4 encode for DSP, while exon 5 encodes for the C-terminus of DSP and the whole DPP. A 4.6-kb RNA transcript was detected on Northern blot analyses of total RNA extracted from immature (open root apices) human teeth using either a human DPP or DSP probe.

Introduction to cariology.

This article examines the discipline of cariology. A brief history is presented, and an overview of the origin and management of caries is discussed. This article frames the picture that the other authors in this issue paint.

Phosphoprotein analysis of sequential extracts of human dentin and the determination of the subsequent remineralization potential of these dentin matrices.

Phosphoprotein appears to play an important role in the mineralization of dentin during tooth development and remineralization after demineralization by dental caries. To better understand this role, we describe the extraction and characterization of phosphoprotein from immature, human root apex dentin during and after EDTA demineralization. The extraction procedure included dissociation of the demineralized dentin matrix by guanidine hydrochloride (Gdn.HCl) followed by subsequent digestion with cyanogen bromide (CNBr) and collagenase. Characterization of these extracts included 'Stains-All' staining of SDS polyacrylamide gels (SDS-PAGE) and amino acid, protein and phosphorus analyses. The ability of these matrices to remineralize was determined by TEM and measuring calcium levels in the remineralized tissue by atomic absorption spectroscopy. The staining of SDS-PAGE gels and amino acid analysis showed that an intact phosphophoryn was extracted from the dentin of the immature apices during EDTA demineralization and that it had an apparent Mr approximately 140,000. In the subsequent extracts and digests, the phosphoprotein has a range of molecular weights, some of which may have been degraded products of the intact phosphoprotein. A greater quantity of phosphoprotein was found in the EDTA-demineralized dentin matrices than in dentin after Gdn.HCl, CNBr and collagenase digests. These EDTA-demineralized matrices also remineralized to a greater extent than those dissociated with Gdn.HCl. The differences in both the quantity and the quality, as defined by the amino acid residue profile, of the phosphoprotein in the sequential extracts of the root apex dentin may be important in affecting the ability of this tissue to remineralize.

Human dentin phosphophoryn nucleotide and amino acid sequence.

Dentin sialoprotein (DSP) and phosphophoryns (DPP) are major dentin-specific non-collagenous proteins and are synthesized by odontoblasts. DPP are extremely acidic, rich in aspartic acid and serine, possess a high affinity for calcium and collagen, and are believed to function in dentin mineralization. Whereas DSP and DPP are the products of a single gene in mouse and rat, an analogous human gene has not been described. Using RT-PCR based cloning strategies, we have cloned human DPP cDNA from immature molar root total RNA. The open reading frame of this human DPP cDNA comprises 2364 bp encoding 788 amino acids rich in serine (58%), aspartic acid (26%) and asparagine (9%). These are mostly arranged as (DSS)n (n = 1-16), DS and NSS motifs. The N-terminal sequence (DDP) matches that obtained from human DPP extracted from the roots of immature teeth. The core protein of this human DPP was calculated to have a molecular weight of 76,906 Da and a net charge of -206 with an isoelectric point of 2.65. Of the serine residues, 53% can potentially be phosphorylated by casein kinases I and II. Thus, this newly cloned human cDNA, which encodes a protein with characteristics similar to rat and mouse DPP, is identified as a human DPP.

Fluoride release and re-uptake in direct tooth colored restorative materials.

OBJECTIVES: Glass ionomers may be "recharged" through topical fluoride (F-) treatments; however, this reported "recharging," may be attributed to surface changes after F- treatment. This study examined differences in F- release and re-uptake among dual-cured and chemically-cured glass ionomers, and a photo-cured F- releasing composite. A secondary goal was to determine if tensile strength or surface roughness changed due to F- release, or F- re-uptake and re-release. METHODS: In Phase 1, initial surface roughness and diametral tensile strength were measured. F- release was measured for 30 days. Strength and roughness were then remeasured. In Phase 2, surface roughness was measured, then materials were treated with a 5000 ppm neutral F- gel, the same gel without F-, or phosphoric acid. F- release was measured for 30 days, then final surface roughness and strength were determined. RESULTS: Significant differences were found in amount and rate of F- release, and F- re-uptake and re-release among study materials and enamel controls (p < 0.001). The amount and rate of F- re-release after NaF treatment differed significantly from F- release after acid treatment in glass ionomers, although both groups showed increased F- release after surface treatment (p < 0.001). There were no significant changes in tensile strength or surface roughness after F- release or F- re-uptake and re-release as determined by ANOVA. SIGNIFICANCE: The results of this in vitro study indicate that applications of neutral 5000 ppm F- gel to aged glass ionomer restorations results in a significant fluoride uptake and subsequent release. The data suggest that the application of neutral fluoride gel to glass ionomer restorations in situ may result in increases in oral fluoride concentrations, without affecting the restoration's surface roughness or tensile strength.

Characterization and identification of a human dentin phosphophoryn.

The present study further characterizes an extract from immature, human tooth apicies from which an intact dentin phosphoprotein has been identified. Third molar apicies from developing roots were decalcified in 10% EDTA until Ca2+ was undetectable in the decalcifying solution. The crude extract was run on 7.5% SDS-PAGE and stained with "Stains-All." Four distinct bands were found and the molecular weights were 140, 60, 50, and 34 k. When run on a SDS-PAGE under nonreducing conditions the 60, 50, and 34 k bands were absent. These results suggest that the lower molecular weight bands may be subunits of the larger protein. The extract was then further purified by adding CaCl2 and MgCl2 to precipitate the phosphoprotein. The precipitate was subjected to a DEAE-Sepharose CL6B column and eluted by 0-0.7 M NaCl gradient solution. The amino acid composition of the purified phosphoprotein was determined and the extract was found to be rich in serine and aspartic acid residues. The N-terminal peptide Asp-Asp-Pro was identified. The sequence of the three amino acids is identical to rat incisor phosphoprotein.